Thrombopoietin (TPO) is a 332-amino acid glycosylated polypeptide which plays a key role in the regulation of megakaryocytopoiesis, the process in which platelets are produced from bone marrow megakaryocytes. See Kuter et al.,Proc. Natl. Acad. Sci. USA 91: 11104–11108 (1994); Barley et al., Cell 77:1117–1124 (1994); Kaushansky et al., Nature 369:568–571 (1994); Wendling et al., Nature 369: 571–574 (1994); and Sauvage et al., Nature 369: 533–538 (1994). IPO is produced in the liver but exerts its main function in the bone marrow, where it stimulates the differentiation of stem cells into megakaryocyte progenitors as well as megakaryocyte proliferation, polyploidization and, ultimately, their fragmentation into circulating platelet bodies. TPO is also the primary regulator of situations involving thrombocytopenia and has been shown in a number of studies to increase platelet counts, increase platelet size, and increase isotope incorporation into platelets of recipient animals. See, e.g., Metcalf Nature 369:519–520 (1994). Specifically, TPO is thought to affect megakaryocytopoiesis in several ways: (1) it produces increases in megakaryocyte size and number; (2) it produces an increase in DNA content, in the form of polyploidy, in megakaryocytes; (3) it increases megakaryocyte endomitosis; (4) it produces increased maturation of megakaryocytes; and (5) it produces an increase in the percentage of precursor cells, in the form of small acetylcholinesterase-positive cells, in the bone marrow.
Platelets are necessary for blood clotting and when their numbers are very low a patient is at risk of death from catastrophic hemorrhage. Thus, TPO has potential useful application in both the diagnosis and the treatment of various hematological disorders, for example, diseases primarily due to platelet defects. Likewise, TPO has potential application in the treatment of thrombocytopenic conditions, especially those derived from chemotherapy, radiation therapy, or bone marrow transplantation as treatment for cancer or lymphoma. Indeed, ongoing clinical trials in cancer patients have shown that recombinant human TPO is effective in decreasing the platelet nadir and enhancing platelet recovery when given with high-dose carboplatin chemotherapy. See Basser Blood, 1997, 89: 3118. Similar results have also been obtained in clinical studies with pegylated megakaryocyte differentiation factor (peg-MGDF, a pegylated truncated N-terminal fragment of human TPO). See e.g., Fanucchi (1997) N Engl J Med, 336: 404.
Because the slow recovery of platelet levels in patients suffering from thrombocytopenia is a serious problem, it would be desirable to provide compounds which allow for the treatment of thrombocytopenia by acting as a TPO mimetic. A few years ago, the development of TPO peptide mimetics was reported (WO 96/4018, WO 96/40750, WO 98/25965). These peptides were designed to bind and activate the TPO-R but have no sequence homology to the natural TPO. In recent years, a number of small-molecule agents with TPO mimetic activity been reported. These include 1,4-benzodiazepin-2-ones (JP11001477), metal complexes derived from Schiff base ligands (WO 99/11262), cyclic polyamine derivatives (WO 00/28987), thiazol-2-yl-benzamides (WO 01/07423, WO 01/53267), azo-aryl derivatives (WO 00/35446, WO 1/17349), 2-aryl-naphthimidazoles (WO 01/39773, WO 01/53267) and semicarbazone derivatives (WO 01/34585). In cell-based systems, all these molecules can activate signal transduction pathways that are dependent on the presence of the TPO receptor on the cell membrane, suggesting some type of direct interaction with the TPO receptor itself. As disclosed herein it has unexpectedly been discovered that certain substituted thiosemicarbazone derivatives are very effective and potent agonists of the TPO receptor. Some of the most preferred compounds of this class were found to stimulate the proliferation and differentiation of TPO-responsive human cell lines and human bone marrow cultures with full TPO efficacy at concentrations below 100 nM.